• Feedstocks Analysed at Celignis

Background on Grass

Grasses can be classified, according to their photosynthetic pathways, as either C3 or C4 plants. C3 grasses that could be considered as energy crops for high-capacity, low-cost production include reed canary grass and giant reed while the corresponding C4 crops include miscanthus and switchgrass. Signifcant quantities of grass biomass are also obtained in municipal areas due to horticultural activities.

Celignis founder Daniel Hayes has considerable experience in the chemical and near-infrared analysis of grasses and has characterised a large number of samples from a wide variety of species.

Components of Grasses

The above ground shoot component of grasses consists of a stem and leaves. The stems are mostly hollow, cylindrical and interrupted at intervals by swollen joints or nodes from which the leaves originate. The parts of the stem between the nodes are termed the internodes.

Rhizomes are a special type of shoot that occur in some grassy species (e.g. miscanthus). They grow underground in a horizontal manner and their internodes are usually short. The only leaves present on rhizomes are small and scale-like. Rhizomes serve as survival organs after periods of senescence and also are often used for storage.

The lower portion of the leaf forms a sheath, which protects the shoots. The second half of the leaf opens out to the leaf blade.

The proportion of biomass components vary with the species. For example, with barley and wheat straw, internodes dominate (59% and 73% of the dry mass, respectively) while leaves (including sheath and blade) constitute an intermediate, and nodes a minor portion of the aboveground dry matter.

Extractives tend to be more prevalent in grasses than woody biomass. In particular, concentrations of water-soluble carbohydrates tend to be relatively high. Glucose and fructose are the two most important free-monosaccharides that occur in grasses and can have dry matter concentrations of approximately 1-3%. More prevalent is the disaccharide sucrose, which can take up 2-8% of the dry matter in forage grasses. In certain grasses, such as perennial ryegrass (Lolium perenne) and cocksfoot (Dactylis glomerata), oligosaccharides such as raffinose and stachyose have been detected, but only in relatively small concentrations. The polysaccharide fructans tend to be the most abundant of the water soluble carbohydrates, constituting around 5-9% of the dry matter of forage grasses, although values as high as 12% have been reported for perennial ryegrass.

Analysis of Grass at Celignis

Celignis Analytical can determine the following properties of Grass samples:

Lignocellulosic Properties of Grass

Cellulose Content of Grass

Cellulose is typically the most abundant constituent, by mass, in most grass species. However, its content can vary significantly according to: the stage of growth; the plant fraction (e.g. node, internode, leaf etc.); and the particular species and variety of grass.

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Hemicellulose Content of Grass

Xylans are the dominant hemicellulose type in most species. As with cellulose, the hemicellulose content of grasses can be highly variable.

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Lignin Content of Grass

The lignin content of grasses is significantly lower than that of wood, although lignin-carbohydrate associations tend to be stronger. There is particular covalent cross-linking (through phenolic acids) between arabinoxylans and lignin polymers. The cuticle contains waxes and waxy polymers that can be also be cross-linked within lignin-like phenolics.

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Starch Content of Grass

The starch content of grasses can be high, when compared with some other biomass feedstocks. It will also vary significantly according to the growth stage of the plant.

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Uronic Acid Content of Grass

Uronic acids can be present as side chains attached to the main backbone of hemicelluloses in grasses. They are typically more abundant in the early-stages of growth. Furthermore, the concentrations of uronic acids tends to be greatest in the nodes, lower in the internodes, and at intermediate levels in the leaves.

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Enzymatic Hydrolysis of Grass

We can undertake tests involving the enzymatic hydrolysis of Grass. In these experiments we can either use a commercial enzyme mix or you can supply your own enzymes. We also offer analysis packages that compare the enzymatic hydrolysis of a pre-treated sample with that of the native original material.

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Bioenergy Properties of Grass

Ash Content of Grass

Grasses tend to have higher ash contents than wood. Regarding the constituent elements, the epidermal cells of some species, particularly cereals, contain high levels of silica.

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Heating (Calorific) Value of Grass

Grasses tend to have lesser heating values than woods, due to their lower lignin and high ash contents. The effective heating value is also greatly dependent on the moisture content of the feedstock, which can vary significantly according to the stage of development of the plant.

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Ash Melting Behaviour of Grass

Ash melting, also known as ash fusion and ash softening, can lead to slagging, fouling and corrosion in boilers which may reduce conversion efficiency. We can determine the ash melting behaviour of Grass using our Carbolite CAF G5 BIO ash melting furnace. It can record the following temperatures:

Ash Shrinkage Starting Temperature (SST) - This occurs when the area of the test piece of Grass ash falls below 95% of the original test piece area.

Ash Deformation Temperature (DT) - The temperature at which the first signs of rounding of the edges of the test piece occurs due to melting.

Ash Hemisphere Temperature (HT) - When the test piece of Grass ash forms a hemisphere (i.e. the height becomes equal to half the base diameter).

Ash Flow Temperature (FT) - The temperature at which the Grass ash is spread out over the supporting tile in a layer, the height of which is half of the test piece at the hemisphere temperature.

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Major and Minor Elements in Grass

Examples of major elements that may be present in Grass include potassium and sodium which are present in biomass ash in the forms of oxides. These can lead to fouling, ash deposition in the convective section of the boiler. Alkali chlorides can also lead to slagging, the fusion and sintering of ash particles which can lead to deposits on boiler tubes and walls.

We can also determine the levels of 13 different minor elements (such as arsenic, copper, and zinc) that may be present in Grass.

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Analysis of Grass for Anaerobic Digestion

Biomethane potential (BMP) of Grass

At Celignis we can provide you with crucial data on feedstock suitability for AD as well as on the composition of process residues. For example, we can determine the biomethane potential (BMP) of Grass. The BMP can be considered to be the experimental theoretical maximum amount of methane produced from a feedstock. We moniotor the volume of biogas produced allowing for a cumulative plot over time, accessed via the Celignis Database. Our BMP packages also involve routine analysis of biogas composition (biomethane, carbon dioxide, hydrogen sulphide, ammonia, oxygen). We also provide detailed analysis of the digestate, the residue that remains after a sample has been digested. Our expertise in lignocellulosic analysis can allow for detailed insight regarding the fate of the different biogenic polymers during digestion.

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Physical Properties of Grass

Bulk Density of Grass

At Celignis we can determine the bulk density of biomass samples, including Grass, according to ISO standard 17828 (2015). This method requires the biomass to be in an appropriate form (chips or powder) for density determination.

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Particle Size of Grass

Our lab is equipped with a Retsch AS 400 sieve shaker. It can accommodate sieves of up to 40 cm diameter, corresponding to a surface area of 1256 square centimetres. This allows us to determine the particle size distribution of a range of samples, including Grass, by following European Standard methods EN 15149- 1:2010 and EN 15149-2:2010.

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Publications on Grass By The Celignis Team

Gottumukkala L.D. Gorgens J.F (2016) Biobutanol production from lignocellulosics, Biofuels Production and future perspectives, Taylor & Francis group

Next-generation biofuels from renewable sources have gained interest among research investigators, industrialists, and governments due to major concerns on the volatility of oil prices, climate change, and depletion of oil reserves. Biobutanol has drawn signicant attention as an alternative transportation fuel due to its superior fuel properties over ethanol. e advantages of butanol are its high energy content, better blending with gasoline, less hydroscopic nature, lower volatility, direct use in convention engines, low corrosiveness, etc. Butanol production through (acetone, butanol, and ethanol) ABE fermentation is a well-established process, but it has several drawbacks like feedstock cost, strain degeneration, product toxicity, and low product concentrations. Lignocellulosic biomass is considered as the most abundant, renewable, low-cost feedstock for biofuels. Production of butanol from lignocellulosic biomass is more complicated due to the recalcitrance of feedstock and inhibitors generated during the pretreatment and hydrolysis process. Advanced fermentation and product recovery techniques are being researched to make biobutanol industrially viable.

Vasudeo Zambare, Archana Zambare, Debmalya Barh, Lew Christopher (2012) Optimization of enzymatic hydrolysis of prairie cordgrass for improved ethanol production, Journal of Renewable and Sustainable Energy 4(3): 1-8


Prairie cordgrass (PCG), Spartina pectinata, is considered an energy crop with potential for bioethanol production in North America. The focus of this study was to optimize enzymatic hydrolysis of PCG at higher solids loadings using a thermostable cellulase of a mutant Penicillium pinophilum ATCC 200401. A three variable, five-level central composite design of response surface methodology (RSM) was employed in a total of 20 experiments to model and evaluate the impact of pH (4.1–6.0), solids loadings (6.6%–23.4%), and enzyme loadings (6.6–23.4 FPU/g dry matter, DM) on glucose yield from a thermo-mechanically extruded PCG. The extruded PCG was first hydrolyzed with the crude P. pinophilum cellulase and then fermented to ethanol with Saccharomyces cerevisiae ATCC 24860. Although all three variables had a significant impact, the enzyme loadings proved the most significant parameter for maximizing the glucose yield. A partial cubic equation could accurately model the response surface of enzymatic hydrolysis as the analysis of variance showed a coefficient of determination (R2) of 0.89. At the optimal conditions of pH of 4.5, solids loadings of 10% and enzyme loadings of 20 FPU/g DM, the enzymatic hydrolysis of pretreated PCG produced a glucose yield of 76.1% from the maximum yield which represents an increase of 15% over the non-optimized controls at the zero-level central points. The predicted results based on the RSM regression model were in good agreement with the actual experimental values. The model can present a rapid means for estimating lignocellulose conversion yields within the selected ranges. Furthermore, statistical optimization of solids and enzyme loadings of enzymatic hydrolysis of biomass may have important implications for reduced capital and operating costs of ethanol production.

Vasudeo Zambare, Archana Zambare, Kasiviswanathan Muthukumarappan, Lew Christopher (2011) Biochemical characterization of thermophilic lignocellulose degrading enzymes and their potential for biomass bioprocessing, International Journal of Energy and Environment 2(1): 99-112


A thermophilic microbial consortium (TMC) producing hydrolytic (cellulolytic and xylanolytic) enzymes was isolated from yard waste compost following enrichment with carboxymethyl cellulose and birchwood xylan. When grown on 5% lignocellulosic substrates (corn stover and prairie cord grass) at 600 C, the thermophilic consortium produced more xylanase (up to 489 U/l on corn stover) than cellulase activity (up to 367 U/l on prairie cord grass). Except for the carboxymethyl cellulose-enriched consortium, thermo-mechanical extrusion pretreatment of these substrates had a positive effect on both activities with up to 13% and 21% increase in the xylanase and cellulase production, respectively. The optimum temperatures of the crude cellulase and xylanase were 600 C and 700 C with half-lives of 15 h and 18 h, respectively, suggesting higher thermostability for the TMC xylanase. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the crude enzyme exhibited protein bands of 25-77 kDa with multiple enzyme activities containing 3 cellulases and 3 xylanases. The substrate specificity declined in the following descending order: avicel>birchwood xylan>microcrystalline cellulose>filter paper>pine wood saw dust>carboxymethyl cellulose. The crude enzyme was 77% more active on insoluble than soluble cellulose. The Km and Vmax values were 36.49 mg/ml and 2.98 U/mg protein on avicel (cellulase), and 22.25 mg/ml and 2.09 U/mg protein, on birchwood xylan (xylanase). A total of 50 TMC isolates were screened for cellulase and xylanase secretion on agar plates. All single isolates showed significantly lower enzyme activities when compared to the thermophilic consortia. This is indicative of the strong synergistic interactions that exist within the thermophilic microbial consortium and enhance its hydrolytic capabilities. It was further demonstrated that the thermostable enzyme-generated lignocellulosic hydrolyzates can be fermented to bioethanol by a recombinant strain of Escherichia coli. This could have important implications in the enzymatic breakdown of lignocellulosic biomass for the establishment of a robust and cost-efficient process for production of cellulosic ethanol. To the best of our knowledge, this work represents the first report in literature on biochemical characterization of lignocellulose-degrading enzymes from a thermophilic microbial consortium.

Vasudeo Zambare, Archana Zambare, Lew Christopher (2011) Enzymatic hydrolysis of hemicellulose from corn stover and prairie cordgrass, Advances in Biotech Research, Microbiologist Society and Education Book Publisher & Distributor
Aswathy U.S, Sukumaran R.K, Devi G.L, Rajasree K.P, Singhania R.R. (2010) Bio-ethanol from water hyacinth biomass: an evaluation of enzymatic saccharification strategy, Bioresource technology 101(3): 925-930

Biomass feedstock having less competition with food crops are desirable for bio-ethanol production and such resources may not be localized geographically. A distributed production strategy is therefore more suitable for feedstock like water hyacinth with a decentralized availability. In this study, we have demonstrated the suitability of this feedstock for production of fermentable sugars using cellulases produced on site. Testing of acid and alkali pretreatment methods indicated that alkali pretreatment was more efficient in making the sample susceptible to enzyme hydrolysis. Cellulase and ?-glucosidase loading and the effect of surfactants were studied and optimized to improve saccharification. Redesigning of enzyme blends resulted in an improvement of saccharification from 57% to 71%. A crude trial on fermentation of the enzymatic hydrolysate using the common baker’s yeast Saccharomyces cerevisiae yielded an ethanol concentration of 4.4 g/L.

Examples of Other Feedstocks Analysed at Celignis